Characterization and Optimization of In Vitro Assay Conditions for (1,3).BETA.-Glucan Synthase Activity from Aspergillus fumigatus and Candida albicans for Enzyme Inhibition Screening.

Abstract

sodium salt, 5mM NaF, 250mM sucrose, and 10 mM NaH2PO4; and for A. fumigatus, 50mM HEPES, 10mM EDTA, 750mM sucrose, 10mM NaH2PO4, 100mM cellobiose and During the last three decades there has been a dramatic increase in the frequency of fungal infections, especially disseminated systemic mycoses in immunodeficient hosts1,2). Mycoses in compromised hosts are mainly the result of opportunistic infections by organisms that are normally harmless, asymptomatic commensals which can be, under certain conditions, pathogenic3,4). Species of Aspergillus, Candida, Coccidioides, Cryptococcus, Histo

Cryptococcus neoformans Resistance to Echinocandins: (1,3)β-Glucan Synthase Activity Is Sensitive to Echinocandins

“…Reports in the literature exist for in vitro (1,3)␤-glucan synthase activity from several fungal organisms (13,16,28,29,40,41,43,48) as well as from C. neoformans (42); however, optimal in vitro assay conditions have not been previously reported for C. neoformans. We have determined the optimal assay conditions for in vitro (1,3)␤-glucan synthase activity from C. neoformans by systematically testing compounds that have been reported to stimulate or stabilize (1,3)␤-glucan synthase activity in vitro (10,13,24,29,41).…”

“…We compared the caspofungin MIC and the K i(app) values for C. neoformans with those of A. fumigatus, S. cerevisiae, and N. crassa. It is important to note that, in each case, we used assay conditions optimum for each fungus (9,40,48). We reasoned that if resistance to caspofungin were due solely to the resistance of the enzyme activity, then we would observe a strong positive correlation between MIC and K i (app) .…”

“…To determine the effects of caspofungin and cilofungin on C. neoformans glucan synthase activity, cells were lysed as described above, and enzyme activity was assayed in the presence of various concentrations of substrate and inhibitor. For Saccharomyces cerevisiae BJ2168 (ATCC 208277), Neurospora crassa (74-OR8-1a), and Aspergillus fumigatus (ATCC 16424), K i(app) values were determined essentially as previously described (9,40,48). Enzyme kinetic data were analyzed using ChemSW Enzyme Kinetics Pro (version 2.36) by SynexChem (Fairfield, CA) for MS Windows 2000.…”

Part I Towards a total synthesis of papulacandin D Part II Towards a total synthesis of amphidinolide X

“…These building blocks would allow us to gain access to complex molecules or their analogues, typified by the prostaglandins (45), palau'amine (46), jatrophane diterpenes (47) and ophiobolins (48). [17][18][19] Apart from these, the range of natural products containing the cyclopentyl moiety spans the alkaloids, steroids, mono-, di-, tri-and sesquiterpenoids and marine natural products.…”

Idenfitication and characterization of Tft1, a glycosyltransferase necessary for cell wall β,3-1,4-Glucan synthesis in Aspergillus fumigatus

“…These genes encode for plasma membrane bound enzymes with high molecular masses and are highly homologous to the single AGS gene found in Schizosaccharomyces pombe 51,52 . A strain lacking all three of these genes shows a complete loss of α-1,3glucan production 51 [55][56][57] . Importantly, deletion of either of the subunits is lethal to the cell 47,57 .…”